The aim of this research is to elucidate the mechanism by which maleylacetoacetate isomerase together with glutathione catalyzes cis-trans isomerization of maleylacetoacetate and maleylacetone. Proposed mechanisms will be tested through the use of substrate analogues designed to trap suggested intermediates in these mechanisms. One mechanism suggests nucleophilic attack by the enzyme on the keto group of the substrate to form a hemiketal, thiohemiketal, or carbinolamine or attack by glutathione on the keto group to form a thiohemiketal. A substrate analogue in which the keto group is replaced by a methyl ester is proposed so that any of the intermediates might eventually result in a trapped acylated enzyme. Another aspect of this mechanism suggests necleophilic attack by GSH or the enzyme at the isomerizing carbon-carbon double bond. The use of the substrate analogue, 4-oxy-6-oxo-2,3-epoxy - 4-hepteneoic acid, where the epoxide has been introduced in place of the double bond, is suggested as a trap for this aspect. Another proposed mechanism, where first electrophilic attack by the enzyme on the substrate's C-5 or carbonyl oxygen at C-6 followed by nucleophilic attack at the isomerizing bond, is also proposed for study. The epoxy substrate analogue would also be trapped were this mechanism to operate. The behavior with both analogues should differentiate between the two mechanisms. Radioactive trapped intermediate analogues would be prepared and the enzyme hydrolyzed to determine the site of attachment. Secondary alpha-deuterium kinetic isotope effects studies are planned to help establish the addition site on the substrate. Studies with GSH analogues will be continued to determine its function and mode of binding to the enzyme. Other mechanisms will be considered.